Neurotransmitters and hormones rely upon G proteins to exert their effects on target cells. In contrast, growth factor tyrosine kinase receptors recruit a cascade of linked signaling proteins including ras-like GTPases to regulate cell growth and differentiation. Recent compelling evidence indicates that additional novel signaling proteins, including certain regulators of G protein signaling (RGS proteins) integrate G protein and ras superfamily-directed pathways at many levels. RGS proteins are highly diverse, multifunctional proteins that bind directly to G proteins to regulate their functions. RGS14 contains domains (RGS, GoLoco and RID domains) that we and others have shown to interact with active Galpha-i /o-GTP (RGS domain), inactive Galpha-i/o-GDP (GoLoco domain) and with the ras-like GTPases rapl and rap2 (RID domain). Activation of rap1/2 pathways in host cells stimulates MAPK/Erk kinase signaling and neuronal differentiation. Roles for RGS14 as a dual regulator of Gai and Gao signaling are not known. Furthermore, how RGS14, Galpha-i/o and rap1/2 interact to regulate one anothers functions, and what cellular mechanisms are involved in regulating these interactions and linked cellular responses is not known. Our preliminary studies indicate that RGS14 is phosphorylated by kinases involved in G protein and rap signaling. Our hypothesis is that RGS14 is an integrator of G protein (Galpha-i/o) and rap-directed signaling pathways, and that stimulus-initiated phosphorylation regulates RGS 14 interactions with Galpha and rap, its subcellular localization, and its signaling functions. Using modern molecular, cellular and biochemical approaches, the specific aims will be to: 1. Determine roles for RGS 14 as a bifunctional regulator of receptor and Galpha-i and Galpha-o signaling in membranes. 2. Define cellular and biochemical mechanisms that regulate RGS 14 membrane recruitment and attachment, subcellular localization, and association with mChoR, Galpha-i/o, and rap1/2 in host B35 cells. 3. Define cellular mechanisms for regulating RGS14 phosphorylation in cells, and roles for phosphorylation on RGS14 interactions with mChoR/Gi/o and rap1/2 in vitro, and linked signaling pathways in B35 cells. 4. Determine the effects of overexpressing RGS14 or eliminating native RGS14 in B35 cells on mChoR/Gi/o and rap1/2- directed signaling events and cellular responses. Impact: These studies will identify previously unrecognized cell signaling roles for RGS proteins, and provide important information about novel mechanisms for cross-talk between G protein signaling pathways and rap/MAPkinase signaling pathways. Findings from these studies will clarify our understanding of the complexity of G protein/RGS protein signaling, and identify potential new targets for therapeutic intervention.